Detection of Eukaryotic cDNA in Differential Display Is Enhanced by the Addition of E. coli RNA

We describe a method to enhance the sensitivity of eukaryotic cDNA detection in differential display (DD). Typically, DD protocols require between 200 and 500 ng RNA for each reverse transcription reaction. The addition of Escherichia coli RNA before reverse transcription of eukaryotic RNA increases the detection of DD patterns more than tenfold. The method broadens the applicability of DD and allows the identification of genes that are differentially expressed when the amount of eukaryotic RNA is limited

Identification and Verification of Differential Display cDNAs Using Gene-Specific Primers and Hybridization Arrays

An accurate and streamlined approach to differential display (DD) band identification and verification is described. To minimize false positives, the strategy avoids the use of impure Northern blot probes obtained from PCR-amplified DD bands. To increase throughput, the cloning of DD bands is replaced by a gene-specific primer approach, and hybridization arrays are used in place of Northern blots. In summary, DD bands obtained with long primers were directly sequenced to allow the design and synthesis of gene-specific primers, which were then used to PCR-amplify homogeneous probes for the verification of expression patterns by hybridization array analysis. Differential expression of 60 of the 63 genes tested was confirmed. Thus, false positives are not inherent to DD. The results demonstrate the power of DD used with hybridization arrays to rapidly generate information on expression patterns of differentially expressed genes

3′-End cDNA Pool Suitable for Differential Display from a Small Number of Cells

We have generated a 3′ cDNA pool from the RNA of only 1000 or fewer cells by reverse transcription (RT) from an extended oligo(dT) primer with a 3′ degenerate base and a second strand primer with four degenerate 3′bases, followed by PCR. Reproducible differential displays (DD) can be made from this essentially inexhaustible source of DNA. The method produced DD patterns that are comparable but not identical in band number and size distribution with those obtained by the original RT-DD technique. Northern blots performed with the excised bands verified altered gene expressions. The data indicate that this 3′-end cDNA pool can supplement current PCRbased methods of expression genetics. This pool of cDNA sequences also provides a reliable source for primer-specific gene amplifications